1. Field of the Invention
The present invention is related to the production of an enzyme aggregate having Improved performance and use characteristics. More particularly, the present invention is related to an enzymatic multimer which comprises monomer units of either homologous or heterologous origin which have been covalently bonded to each other. The enzymatic multimer of the invention has advantages such as improved performance in terms of activity, allergenicity or enzyme-substrate interactions.
2. State of the Art
Multiple enzyme aggregates have been suggested for decreasing the allergenicity of the component enzyme(s) by increasing their size. For example, PCT Publication No. 94/10191 discloses oligomeric proteins which display lower allergenicity than the monomeric parent protein and proposes several general techniques for increasing the size of the parent enzyme. Additionally, enzyme aggregates have shown improved characteristics under isolated circumstances. For example, Naka et al., Chem. Lett., vol. 8, pp. 1303-1306 (1991) discloses a horseradish peroxidase aggregate prepared by forming a block copolymer via a 2-stage block copolymerization between 2-butyl-2-oxazoline and 2-methyl-2-oxazoline. The aggregate had over 200 times more activity in water saturated chloroform than did the native enzyme.
Cross-linked enzymes prepared by the addition of glutaraldehyde has been suggested as a means of stabilizing enzymes. However, cross-linking often leads to losses in activity compared to native enzyme. For example, Khare et al., Biotechnol. Bioeng., vol. 35, no. 1, pp. 94-98 (1990) disclose an aggregate of E. coli β-galactosidase produced with glutaraldehyde. The enzyme aggregate, while showing improvement in thermal stability at 55° C., had an activity of only 70.8% of that of the native enzyme which was, however, considered a good retention of activity after cross-linking.
As is understood from above, several alternatives have been developed by those of skill in the art seeking to produce aggregated enzymes for the purpose of decreasing allergenicity or altering activity parameters. However, a problem common to each of these processes is that, when preparing aggregated enzymes according to these prior art teachings, it is not believed feasible to predict how certain enzymes will behave in the aggregated form. Moreover, the formation of an enzyme aggregate according to these prior art teachings is an inexact science which is highly dependent on fortuity, thus presenting a significant barrier to the preparation of a multienzyme aggregate having pre-selected activities.
To overcome these problems, researchers have developed enzymatic aggregates which comprise predetermined fusion proteins. In a typical fusion protein, the gene for one protein is fused to the gene for a second protein and the resultant combination enzyme is expressed as an integral unit. Such fusion proteins, as applied to enzymes, while providing an important advantage in terms of providing a single protein combining multiple enzymatic activities, are problematic in terms of expression and/or secretion in a suitable host cell. For example, proteolytic cleavage, improper folding and secretion problems within the cell resulting from or due to size or tertiary structure represent significant drawbacks of fusion enzyme technology.
Accordingly, it would be desirable to develop a new means of preparing multiple enzyme systems useful for medical, diagnostic or industrial purposes which is capable of being customized in terms of included enzymatic activities and positional interrelationships of those enzymes so as to maximize the kinetics of the specific application. It would further be desirable to develop a new means of preparing multiple enzyme systems which do not have problems in expression and secretion characteristic of fusion proteins, and which allow flexibility in determining the conformation of the resulting multiple enzyme. However, the prior art fails to provide a means for producing a multiple enzyme system having such characteristics.